scholarly journals Characterization of an Active Partition System for the Enterococcus faecalis Pheromone-Responding Plasmid pAD1

2007 ◽  
Vol 189 (23) ◽  
pp. 8546-8555 ◽  
Author(s):  
Maria Victoria Francia ◽  
Keith E. Weaver ◽  
Patricia Goicoechea ◽  
Patricia Tille ◽  
Don B. Clewell
Keyword(s):  
Enterococcus Faecalis ◽  
Consensus Sequence ◽  
Protein A ◽  
Mobility Shift ◽  
Mating Response ◽  
Partition System ◽  
Dnase I Footprinting ◽  
Low Copy Number ◽  
Replication Initiator

ABSTRACT Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol. 175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol. 186:5003-5016, 2004). The present study focuses on the adjacent determinants repB and repC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribed repA and repB. Through mutagenesis and trans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active in trans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of the repBC locus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Functional Analysis of TraA, the Sex Pheromone Receptor Encoded by pPD1, in a Promoter Region Essential for the Mating Response in Enterococcus faecalis

2002 ◽  
Vol 184 (22) ◽  
pp. 6343-6350 ◽  
Author(s):  
Takaaki Horii ◽  
Hiromichi Nagasawa ◽  
Jiro Nakayama
Keyword(s):  
Sex Pheromone ◽  
Enterococcus Faecalis ◽  
Intergenic Region ◽  
Recipient Cell ◽  
Donor Cell ◽  
Northern Analysis ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
Mating Response ◽  
Gel Mobility Shift

ABSTRACT Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response.

scholarly journals Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014 ◽  
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Characterization of a telomere-binding protein from Physarum polycephalum.

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290 ◽  
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals The local transcriptional regulators SacR1 and SacR2 act as repressors of fructooligosaccharides metabolism in Lactobacillus plantarum

2020 ◽  
Author(s):  
Chen Chen ◽  
Linlin Wang ◽  
Haiyan Yu ◽  
huaixiang tian
Keyword(s):  
Lactobacillus Plantarum ◽  
Consensus Sequence ◽  
Protein A ◽  
Pcr Analysis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Global And Local ◽  
Electrophoretic Mobility Shift Assays

Abstract Background: In Lactobacillus plantarum , fructooligosaccharides (FOS) metabolism is controlled by both global and local regulatory mechanisms. Although catabolite control protein A has been identified as a global regulator of FOS metabolism, the functions of local regulators remain unclear. This study aimed to elucidate the roles of two local regulators, SacR1 and SacR2, in the regulation of FOS metabolism in L. plantarum both in vitro and in vivo . Results: The inactivation of sacR1 and sacR2 affected the growth and production of metabolites for strains grown on FOS or glucose, respectively. A reverse transcription-quantitative PCR analysis of one wild-type and two mutant strains ( ΔsacR1 and ΔsacR2 ) of L. plantarum identified SacR1 and SacR2 as repressors of genes relevant to FOS metabolism in the absence of FOS, and these genes could be induced or derepressed by the addition of FOS. The analysis predicted four potential transcription factor binding sites (TFBSs) in the putative promoter regions of two FOS-related clusters. The binding of SacR1 and SacR2 to these TFBSs both in vitro and in vivo was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation, respectively. A consensus sequence of WNNNNNAACGNNTTNNNNNW was deduced for the TFBSs of SacR1 and SacR2. Conclusion: Our results identified SacR1 and SacR2 as local repressors for FOS metabolism in L. plantarum . The regulation is achieved by the binding of SacR1 and SacR2 to TFBSs in the promoter regions of FOS-related clusters. The results provide new insights into the complex network regulating oligosaccharide metabolism by lactic acid bacteria .

Characterization of a telomere-binding protein from Physarum polycephalum

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals The Response Regulator CroR Modulates Expression of the Secreted Stress-Induced SalB Protein in Enterococcus faecalis

2006 ◽  
Vol 188 (7) ◽  
pp. 2636-2645 ◽  
Author(s):  
Cécile Muller ◽  
Yoann Le Breton ◽  
Thierry Morin ◽  
Abdellah Benachour ◽  
Yanick Auffray ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Promoter Region ◽  
Response Regulator ◽  
Signal Transduction System ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Enterococcus faecalis two-component signal transduction system CroRS, also referred as the RR-HK05 pair, is required for intrinsic β-lactam resistance (Y. R. Comenge, R. Quintiliani, Jr., L. Li, L. Dubost, J. P. Brouard, J. E. Hugonnet, and M. Arthur, J. Bacteriol. 185:7184-7192, 2003) and is also suspected to be involved in the expression of salB (previously referred to as sagA), a gene important for resistance to environmental stress and cell morphology (Y. Le Breton, G. Boël, A. Benachour, H. Prévost, Y. Auffray, and A. Rincé, Environ. Microbiol. 5:329-337, 2003). In this report, we provide genetic and biochemical evidence that salB encodes a secreted protein that is expressed from a monocistronic stress-inducible operon. Consistent with CroR being a direct transcriptional activator of the salB expression, CroR was found to bind to the salB promoter region in electrophoretic mobility shift assays. Interestingly, we provide evidence that SalB does not play a role in the intrinsic β-lactam resistance associated with CroRS. We also show that the CroRS system is able to regulate its own expression. The sequence of the CroRS binding site in the salB and croR promoter regions was determined using DNase I footprinting assays.

scholarly journals Characterization of the Organic Hydroperoxide Resistance System of Brucella abortus 2308

2012 ◽  
Vol 194 (18) ◽  
pp. 5065-5072 ◽  
Author(s):  
Clayton C. Caswell ◽  
John E. Baumgartner ◽  
Daniel W. Martin ◽  
R. Martin Roop
Keyword(s):  
Brucella Abortus ◽  
Sequence Similarity ◽  
Mobility Shift ◽  
Organic Hydroperoxide ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Organic Hydroperoxides ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTThe organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression ofohris often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in theBrucella abortus2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenicohrandohrRmutants andlacZpromoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, inB. abortus2308 and that OhrR represses the transcription of bothohrandohrRin this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region betweenohrandohrRthat shares extensive nucleotide sequence similarity with so-called “OhrR boxes” described in other bacteria. While Ohr plays a prominent role in protectingB. abortus2308 from organic hydroperoxide stress inin vitroassays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.

Characterization of the Murine Coagulation Factor X Promoter

2000 ◽  
Vol 84 (12) ◽  
pp. 1031-1038 ◽  
Author(s):  
Julie Wilberding ◽  
Francis Castellino
Keyword(s):  
Coagulation Factor ◽  
Proximal Promoter ◽  
Factor X ◽  
Wild Type ◽  
Gata Factor ◽  
Mobility Shift ◽  
Translational Initiation ◽  
Dnase I Footprinting ◽  
Shift Analysis

SummaryFactor X (FX) is a vitamin K-dependent serine protease zymogen that functions in both the extrinsic and intrinsic pathways of blood coagulation. In this study, the 5’-flanking region of the murine FX gene was analyzed to determine those elements that govern its transcriptional activity and regulation. Consistent with other TATA-less promoters, murine FX contains two start sites of transcription, at bp −5 and −21 relative to the ATG translational initiation codon. The mRNA of FX was found in a number of tissues, including the liver, stomach, intestine, kidney, ovary, testes, spleen, skeletal muscle, and lung. Using DNase I footprinting, three areas of protection have been identified in the proximal 287 bp of the promoter, spanning bp −28 to −218. Further examination of this region revealed transcription factor binding sites for NF-Y, HNF-4, and a GATA factor. Electrophoretic mobility shift analysis (EMSA) confirmed the identities of NF-Y, HNF-4, and GATA-4, all of which were found by transient transfection analyses in HepG2 cells to influence the activity of the promoter. Ablation of the NF-Y site was most dramatic, reducing activity to 10% of that of the wild-type construct. Deletion of the HNF-4 site led to an activity of 25% of wild-type, and a GATA-4 mutation reduced activity to 63% of wild-type values. This investigation revealed the identity of the factors bound at the proximal promoter of the FX gene, and the relative importance of each. This is the first report of a member of the GATA family of transcription factors being important in the regulation of a coagulation-based gene.

scholarly journals The local transcriptional regulators SacR1 and SacR2 act as repressors of fructooligosaccharides metabolism in Lactobacillus plantarum

2020 ◽  
Author(s):  
Chen Chen ◽  
Linlin Wang ◽  
Haiyan Yu ◽  
huaixiang tian
Keyword(s):  
Lactobacillus Plantarum ◽  
Consensus Sequence ◽  
Protein A ◽  
Pcr Analysis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Global And Local ◽  
Electrophoretic Mobility Shift Assays

Abstract Background In Lactobacillus plantarum, fructooligosaccharides (FOS) metabolism is controlled by both global and local regulatory mechanisms. Although catabolite control protein A has been identified as a global regulator of FOS metabolism, the functions of local regulators remain unclear. This study aimed to elucidate the roles of two local regulators, SacR1 and SacR2, in the regulation of FOS metabolism in L. plantarum both in vitro and in vivo. Results A reverse transcription-quantitative PCR analysis of one wild-type and two mutant strains ( ΔsacR1 and ΔsacR2 ) of L. plantarum grown on FOS and ribose identified SacR1 and SacR2 as repressors of FOS metabolism in the absence of FOS. Moreover, genes relevant to FOS metabolism could be induced or derepressed by the addition of FOS. The analysis predicted four potential SacR1 and SacR2 transcription factor binding sites (TFBS) in the putative promoter regions of two FOS-related clusters. The binding of SacR1 and SacR2 to these TFBSs both in vitro and in vivo was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation, respectively. A consensus sequence of WNNNNNAACGNNTTNNNNNW was deduced for the TFBS of SacR1 and SacR2. Conclusion Our results identified SacR1 and SacR2 as local regulators that repress FOS utilization by L. plantarum by binding to TFBSs in the promoter regions of FOS-related clusters. The results provide new insights into the complex network regulating oligosaccharide metabolism by lactic acid bacteria.

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